Abstract 3402: Number of variants of unknown significance (VUS) by next generation sequencing (NGS) and response to systemic therapies in advanced urothelial carcinoma (aUC)

Abstract Background: NGS is routinely used in aUC to identify clinically actionable alterations. NGS assays tier variants by the likelihood of clinical significance, reporting both pathogenic variants and VUS. The number of VUS may serve as a rough estimate for neoantigen load, e.g. tumor mutational burden (TMB). We hypothesized that the number of VUS may represent an easy-to-use biomarker associated with response and overall survival (OS) with certain therapies e.g. immune checkpoint inhibitors (ICI). Methods: We conducted a retrospective study from patients (pts) from a single academic center with aUC and available NGS data derived from FoundationCDx using tumor tissue who had received ICI, enfortumab-vedotin (EV), and/or sacituzumab govitecan (SG) in the first line (1L) or as salvage (2L+). Patients at or above the median were defined as VUS high (VUS-H); those below were VUS low (VUS-L). We analyzed observed response rate (ORR) and OS measured from each therapy start between groups. Cox proportional hazards test was used to compare OS; Fisher’s exact test was used to compare ORR. Kaplan-Meier method was used to calculate survival curves. Results: 90 pts had available NGS data and received ICI, EV, and/or SG [VUS-H (≥9 VUS) N = 51; VUS-L (0-8 VUS) N = 39]. VUS was correlated with TMB (Pearson coefficient = 0.78). Baseline characteristics including age, sex, ECOG PS, prior extirpative surgery rate, and primary tumor site were similar between groups, except for presence of liver metastases at diagnosis (VUS-H = 10%, VUS-L = 36%; p= 0.003). Results are described in the table. Conclusions: Higher number of VUS detected by tissue-based NGS was associated with higher ORR and OS with 1st line ICI. Limitations include retrospective nature, lack of randomization, moderate sample size and power, selection and confounding biases. Results are hypothesis-generating and need external validation. Table: ORR and OS analyses by group N ORR % OR (95%CI) p mOS, months(95%CI) HR(95%CI) p 1L ICI VUS-H 23 60.9%(14/23) 7.78(1.74 – 34.71) 0.009 39(24-NR) 0.30(0.14, 0.66) 0.003 VUS-L 18 14.3%(3/18) 8.0(5.3-29) 2+L ICI VUS-H 16 18.8%(3/16) 1.15(0.16 – 8.27) 1.0 4.3(3.0-24) 1.74(0.73 – 4.12) 0.2 VUS-L 12 18.2%(2/12) 11(6.7-NR) 2+L EV VUS-H 20 45.0%(9/20) 2.86(0.47 – 17.35) 0.41 26(12-NR) 0.63(0.24, 1.62) 0.3 VUS-L 9 22.2%(2/9) 10(8.8-NR) 2L + SG VUS-H 13 7.7%(1/13) undefined 1.0 10(6.1-NR) 0.35(0.11 – 1.13) 0.08 VUS-L 9 0.0%(0/9) 7.1(2.5-NR) Citation Format: Thomas L. Enright, Jacob B. Leary, Dimitra R. Bakaloudi, Rafee Talukder, Michael T. Schweizer, Jessica E. Hawley, Andrew C. Hsieh, Todd Yezesfski, Bruce Montgomery, Evan Y. Yu, Petros Grivas, Ruben Raychaudhuri, Ali R. Khaki. Number of variants of unknown significance (VUS) by next generation sequencing (NGS) and response to systemic therapies in advanced urothelial carcinoma (aUC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3402.